Proper protein folding is a key issue in maintaining healthy neurons, including the photoreceptor cells of the retina. Stresses on the photoreceptor proteome may be caused by environmental factors such as light damage or by genetic factors such as misfolding mutations in rhodopsin and many other photoreceptor proteins. The proteome is maintained by a class of proteins called molecular chaperones. These chaperones protect proteins from aggregation, channel their folding pathways and facilitate their association into multi-protein assemblies. An important type of molecular chaperone is the type II chaperonin found in the eukaryotic cytosol, termed CCT (Cytosolic Chaperonin containing Tailless complex polypeptide 1, also called TRiC). The number of proteins known to require CCT for their folding is in the hundreds and continues to grow. Among these are the G protein ? subunits (G?) which form the G?? and G?5-RGS (Regulator of G protein Signaling) dimers that are key components in visual signaling. Both G?? and RGS-G?5 also require the CCT co-chaperone, phosducin-like protein, for proper folding and dimer formation. In previous work, we have determined the structures of two intermediates in G?? assembly by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and site-specific cross-linking with unnatural amino acids. These structures have provided molecular detail into the mechanism of G?? assembly. In Aim 1, we propose to determine the structures of similar intermediates in RGS-G?5 assembly to understand at the molecular level how CCT and PhLP1 assist in RGS-G?5 dimer formation. An additional role for CCT in Bardet-Biedl syndrome (BBS) has also been demonstrated. BBS is a genetic disease of ciliary dysfunction characterized by multiple pathological conditions including retinal degeneration. BBS is caused by an inability to form the BBSome, a complex of eight proteins that is essential for vesicle trafficking to cilia. CCT and a CCT-like complex made up of three proteins whose mutations are known to cause BBS (BBS 6, 10 and 12) are required for the assembly of the BBSome. In Aim 2, we propose similar structural studies to determine the molecular mechanism of BBSome assembly and BBSome function. The information gained from these studies will be vital in designing chaperone-based methods to treat retinal diseases caused by RGS-G?5 and BBSome malfunctions.